The SARS-Coronavirus (SARS-HCoV) has been implicated as the causative agent of SARS (severe acute respiratory syndrome) in humans. This virus has caused multiple deaths in various affected countries throughout the world. The SARS coronavirus spike protein has only 30% identity at the amino acid level to the spike proteins of the previously characterised coronaviruses. Recently, the genome of SARS isolates implicated in the 2003 Toronto outbreak were sequenced in their entirety (Marco et al., 2003, Science 300: 1399-1404; Rota et al., 2003, Science 300: 1394-1399). The production of mAbs specific to this agent is critical for diagnostic development, vaccine research and studies of viral pathogenesis. Assays that detect the presence of virally encoded proteins or nucleic acids may be preferable for diagnosis of SARS infections as the development of serum antibodies is quite protracted (Li et al., 2003, N. Engl. J. Med. 349: 508-509).
Coronaviruses acre enveloped, single stranded RNA viruses that replicate in the host cell cytoplasm [Fields, B. N., Knipe, D. M., Howley, P. M., and Griffin, D. E. (2001) Fields Virology (Lippincott Williams & Wilkins, Philadelphia, ed. 4)]. The coronaviruses form a single genus of the family Coronaviridae and the virions are large (80-160 nm in diameter), pleomorphic but generally spherical particles. Virions of most coronaviruses contain three major proteins: the phosphorylated nucleocapsid protein N; a small membrane-embedded glycoprotein (M); and a large club-shaped peplomer glycoprotein (S) which appears in EM micrographs as protruding spikes 20 nm in length. The M protein is synthesized on ribosomes bound to the endoplasmic reticulum and accumulates in the Golgi apparatus. The subcellular localization of M protein to the Golgi is believed to determine the site of virus budding from the infected cell. The S protein mediates many of the biological properties of the virus, including attachment to cell receptors, penetration, and cell-fusion, and it is the major target for virus-neutralizing antibodies (Collins et al., 1982, Virology 61:1814-1820; Talbot et al., 1984 Virology 132: 250-260; Wege and Dorrier, 1984, J. Gen. Virol. 65: 217-227; Laude et al., 1986, J. Gen. Virol. 67: 119-130; Jimenez et al., 1986, J. Virol. 60: 131-139; Godet et al., 1994, J. Virol. 68: 8008-8016). A proportion of the S glycoprotein that is not incorporated into budding virions is transported to the plasma membrane of the cell where it remains bound to the cell surface (Gerna et al., 1982, J. Gen. Virol. 60: 385-390).
Coronaviruses infect a wide range of mammalian hosts to produce a variety of disease outcomes including respiratory disease, enteritis and encephalitis. Antigenic similarities between various coronaviruses have been demonstrated to reside in the S protein and have been used to study evolution of this virus family [Brian, D. A., Hogue, B., Lapps, W., Potts, B. and Kapke, P. (1983) Proc. 4th Int. Symp. Neonatal Diarrhea (S.D. Acres, Saskatoon, Canada ed.)]. For most coronaviruses causing enteric and respiratory diseases the pathophysiological events leading to clinical symptoms are due to the acute cytocidal infection of the target cells. These infections can be limited by the local immune response resulting in the production of secretory antibodies specific for the S protein (Enjuanes et al., 1995, Dev. Biol. Stand. 84: 145-152). In contrast, many coronaviruses are maintained and spread in the population as inapparent and subclinical infections. The sequence of events leading to chronic disease is unknown but likely depends on the expression of viral genes, the functional impairment of host cells and the interaction with the host immune response.
There is a critical need to elucidate the immunologic basis for protection against SARS virus. The immunogenetics of antibody responses to protective epitopes is of particular importance and will lead to a clearer understanding of the nature of protective antibody responses to SARS. Lastly, the production of protective monoclonal antibodies may lead to the development of new recombinant therapeutic antibodies in order to provide rapid protection in SARS patients. In the present work we describe the development of murine mAbs against the SARS HCoV involved in the Toronto SARS outbreak. The mAbs were analysed for pertinent immunochemical properties and for their ability to neutralize the SARS virus in vitro.